Many adult human illnesses including Alzheimer's disease (AD), and Parkinson's disease and amyotrophic lateral sclerosis (ALS) involve pathologic change of neurons, which results in their loss through apoptosis. The long-term objective of this research in our laboratory is to understand how signal-controlled intracellular mechanisms regulate neuronal survival and apoptosis during development and neurodegeneration. Studies have shown that cyclin dependent kinase 5 (Cdk5) plays a key role in the apoptosis of mature neurons. Our recent findings suggest that Cdk5 functions in the nucleus to regulate apoptosis. We propose in the present application to identify novel nuclear mechanisms by which Cdk5 induces neuronal apoptosis. Our specific aims are: 1. to identify and characterize novel regulatory targets of Cdk5 in neuronal nucleus; 2. to explore regulation of p38 MAPK signaling pathway by Cdk5 in neurons; and 3. to identify and characterize additional Cdk5 regulatory sites in MEF2 (myocyte enhancer factor 2). To identify novel nuclear substrates of Cdk5, we will use Cdk5 binding assays to first isolate proteins from nuclear preparations of primary neurons and reveal their identify by Mass phosphorylation by mutagenesis. We will assess how Cdk5-mediated phosphorylation of p38 MAPK affects its subcellular localization and Spectrometer analysis. We will then confirm them as Cdk5 substrates in biochemical and biological assays. To explore whether Cdk5 directly regulates p38 MAPK pathway, we will test if p38 MAPK is a substrate of Cdk5 by in vitro kinase assays and in vivo phosphorylation studies. We will determine sites of regulation of its downstream targets. We will test Cdk5-mediated phosphorylation of p38 MAPK in models of neurotoxin-induced apoptosis. We will also explore whether Cdk5 directly regulates p38 MAPK upstream activators. Additional regulatory sites in various isoforms of MEF2 will be identified by mutagenesis. The potential effects of Cdk5-mediated phosphorylation of alternatively spliced MEF2C variants will be assessed using MEF2C mutants in reporter gene activation assays and neuronal survival assays. Identifying these novel nuclear regulatory targets of Cdk5 will allow us to explore the mechanisms by which Cdk5 regulates the survival machinery in the nucleus. This should further our understanding of the molecular process of neurotoxin-induced apoptosis, which underlies the pathogenesis of many neurodegenerative diseases [unreadable] [unreadable]